Development of broad‐spectrum bacterial blight resistance into thermo‐sensitive genic male sterile lines

Two‐line hybrid rice technology is an effective way to increase rice production and improve rice quality. In this study, three bacterial blight (BB) resistance genes, Xa7, Xa21 and Xa23, were introgressed separately into C815S, a popular thermo‐sensitive genic male sterile (TGMS) line to develop five BB‐resistant lines (Hua1005S, Hua1002S, Hua1009S, Hua1006S and Hua1001S) to be resistant against seven races of Xanthomonas oryzae pv. oryzae (Xoo). The two‐line hybrids with heterozygous Xa23 were resistant against seven Xoo strains. But, the hybrids with heterozygous loci for both Xa7 and Xa21 were only resistant against three Xoo strains and were moderately susceptible to the other four strains indicating the role of modifiers influencing the poor expression of dominant BB resistance genes under heterozygous state. Among them, Hua1006S was found to be a promising TGMS line with its higher degree of disease resistance level on account of broad‐spectrum resistance gene Xa23 besides possessing better plant type and rice grain quality features.     

Pyramiding of insect- and disease-resistance genes into an elite indica, cytoplasm male sterile restorer line of rice,'Minghui 63'

The wild‐rice‐derived dominant gene Xa21 conferring multi‐race resistance to bacterial blight and a fused Bt gene cry1Ab/cry1Ac conferring resistance to lepidopteran insects were individually introduced into the same genetic background of an elite indica cytoplasm male sterile (CMS) restorer line ‘Minghui 63′. The line showed the desirable insect‐ and disease‐resistant phenotypes. To maximize the effect, the two genes were also pyramided into the same recipient plant of ‘Minghui 63’ by marker‐assisted selection. After being subjected to natural infestation of leaf‐folders and yellow stem borers and inoculation of Xoo strain mixtures, the pyramiding line and its derived hybrids showed high levels of resistance against both insect damage and disease. Furthermore, data from field trials demonstrated that the hybrids made by crossing this pyramiding line with the CMS lines ‘Zhenshan 97A’ and ‘Maxie A’ retained a similar level of yield under conditions without chemical spray, indicating that the pyramiding genes have a yield‐stabilizing effect on the recipient line and its hybrids.     

不同Xa21转基因杂交稻组合的大田试验与分析

在北京、四川和江苏等地对Xa21转基因杂交稻进行了大田释放实验. 分别对转基因纯合的3个恢复系明恢63-Xa21、盐恢559-Xa21和C418-Xa21与各种不育系配组的23个杂交组合进行了抗性和农艺性状分析. 在不同的杂合遗传背景下转基因Xa21稳定遗传和高效表达, 所有杂交组合均具有对白叶枯病的广谱抗性. 转基因恢复系配制的杂交组合与     

Identification and gene cloning of a male‐sterile mutant Oswbc11 in rice

Commercial application of nuclear genetic male sterility has been improved recently by a novel technique, Seed Production Technology (SPT), which incorporates transgenic maintainer lines capable of propagating non‐transgenic nuclear male‐sterile lines for use as female parents in hybrid production. Here, we identified a rice nuclear male‐sterile mutant, Oswbc11, with abnormal pollen development and lipid transport. We finely mapped the Oswbc11 gene into a 12.5‐kb region on chromosome 10 and found one candidate gene, which had a base substitution (C to T) resulting in a premature stop codon and was functionally confirmed by CRISPR/Cas9 technology. Gene OsWBC11 encodes a sub‐family member of adenosine triphosphate‐binding cassette transporter, which participates in the active transport of a wide range of molecules across membranes. Moreover, the agronomic traits of Oswbc11 mutants showed no significant differences compared to the wild‐type control except for the seed setting rate. These results indicated that Oswbc11 gene could be used in rice hybrid breeding as a recessive nuclear male‐sterile gene combined with CRISPR/Cas9 and the SPT technology and applied in different rice varieties.     

Marker‐assisted introgression of bacterial blight and blast resistance into IR 58025B,an elite maintainer line of rice

IR 58025A is a very popular wild‐abortive cytoplasmic male sterile (WA‐CMS) line of rice and is extensively used for hybrid rice breeding. However, IR 58025A and many hybrids derived from it possess mild aroma (undesirable in some parts of India) and are highly susceptible to bacterial blight (BB) and blast diseases. To improve IR 58025A for BB and blast resistance, we have introgressed a major dominant gene conferring resistance against BB (i.e. Xa21) and blast (i.e. Pi54) into IR 58025B, the maintainer line of IR 58025A. An introgression line of Samba Mahsuri (i.e. SM2154) possessing Xa21 and Pi54 genes in homozygous condition and fine‐grain type was used as donor parent, and backcross breeding strategy was adopted for targeted introgression of the resistance genes. PCR‐based molecular markers tightly linked to Xa21 and Pi54 were used for selection of BB‐ and blast‐resistant lines, while closely linked markers were used for identification of backcross‐derived plants devoid of Rf4 and aroma. At BC₂F₅, four backcross‐derived lines possessing resistance against BB and blast, devoid of aroma, high yield, short plant stature, long‐slender grain type and with recurrent parent genome recovery ranging from 88.8% to 98.6% were selected and advanced for further evaluation. The improved versions of IR 58025B, viz. SB54‐11‐143‐9‐44‐5, SB54‐11‐143‐9‐44‐98, SB54‐11‐143‐9‐44‐111 and SB54‐11‐143‐9‐44‐171, behaved as perfect maintainers when test‐crossed with WA‐CMS lines. Agronomically superior lines of improved IR 58025B are being converted to CMS line through backcrossing for developing high‐yielding and biotic stress‐resistant rice hybrids.     

Out-crossing frequency and genetic analysis of hybrids between transgenic glufosinate herbicide-resistant rice and the weed, red rice

The potential of transferring herbicide resistance from transgenic rice (Oryza sativa L.) varieties to sexually compatible weeds is of paramount importance for development of effective weed control strategies. The objective of this research was to determine the genetic control and frequency of natural outcrossing between a transgenic, glufosinate-resistant rice line and a Louisiana biotype of red rice (Oryza sativa L.). Molecular and phenotypic data showed that outcrossing in field plots between a non-transgenic purple marker line and red rice did occur within one field season, but at a low rate of <1%. Similarly, molecular and phenotypic data demonstrated that glufosinate resistance was transferred from the transgenic line to the red rice biotype in the field within one year at a low frequency of 0.3%. Compared to parental lines, the transgenic-red rice hybrids were extremely late, tall, and never set seed during the normal field season. Genetic analyses in all F₂ populations showed glufosinate resistance behaved in a Mendelian fashion as a single, dominant gene. Presence of the bar gene for glufosinate resistance did not increase fitness or seed fecundity in hybrids or subsequent progeny. The genetic analyses and outcrossing results from this study suggest that an effective management program can be developed to prolong the usefulness of transgenic, glufosinate herbicide technology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide

In this paper, we described the breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer (SSB) and herbicide by conventional crossing of two transgenic parental lines transformed independently with different genes. Two stable transgenic rice lines used as donor parents were developed, one was Zhongguo91 which contained cry1Ab gene (for insect resistance) and bar gene (for tolerance of herbicide), and the other was Yujing6 which contained Xa21 gene (resistance to bacterial blight). The elite restorer line Hui773 was used as recipient and crossed with the two stable transgenic rice lines. Then five successive backcrosses were made using Hui773 as recurrent parent. Two rice elite restorers, T773-1 expressing cry1Ab and bar genes and T773-2 expressing Xa21 gene, were obtained, which were confirmed by PCR analysis and testing selectable marker genes in the hybrid progenies. The cross was made between T773-1 and T773-2 to select stable restorer line carrying Xa21, cry1Ab and bar genes. Finally, we obtained transgenic restorer line T773 with good agronomic traits and obvious multiple resistance to Xanthomonas oryzae pv. oryzae, striped stem borer (Chilo suppressalis) and herbicide. The hybrid F₁ generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis. Our results indicate that it is feasible to develop transgenic hybrid rice cultivar through breeding transgenic restorer lines.     

Marker‐assisted breeding of Pi‐1 and Piz‐5 genes imparting resistance to rice blast in PRR78, restorer line of Pusa RH‐10 Basmati rice hybrid

Rice blast, caused by fungus Magnaporthe grisea, is a serious disease causing considerable economic damage worldwide. Best way to overcome disease is to breed for disease‐resistant cultivars/parental lines of hybrids. Pusa RH10, first aromatic, fine‐grain rice hybrid released and cultivated extensively in India. Hybrid and its parental lines, Pusa 6A and PRR78, are highly susceptible to blast. CO39 pyramid carrying two dominant, broad‐spectrum blast‐resistance genes, viz. Pi‐1 and Piz‐5, used as a donor parent to introgress these genes into PRR78 using marker‐assisted backcrossing (MABC). Microsatellite markers RM5926 and AP5659‐5 tightly linked to Pi‐1 and Piz‐5 genes, respectively, were used for foreground selection to derive introgression lines. Further, these lines were evaluated for agronomic performance, disease reaction and cooking quality traits along with PRR78. Most of the improved lines were on par with PRR78 for all traits evaluated except gelatinization temperature. Recurrent parent genome percentage (RPG) study also revealed similarity of these lines with PRR78. Hybrids derived using improved PRR78 lines were superior over Pusa RH10 in terms of yield.     

Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

Construction of chromosomal segment substitution lines and genetic dissection of introgressed segments associated with yield determination in the parents of a super‐hybrid rice

Heterosis is a phenomenon whereby hybrids of inbred lines produce favourable phenotypes that exceed those of their parents. Traits of interest are higher yield and stronger stress tolerance. The two‐line super‐hybrid rice ‘Liangyoupei9’ (LYP9) shows superiority to both its elite inbred line ‘93‐11’ and ‘Pei'ai64s’ (‘PA64s’) parents and conventional hybrids. However, the genetic basis of its hybrid vigour, especially yield determination, remains elusive. In the present study, a set of 156 chromosome segment substitution lines (CSSLs) carrying overlapping segments from ‘PA64s’ in a genetic background of ‘93‐11’ were constructed and planted in six environments. Three major agronomic traits, viz. panicle length (PL), heading date (HD) and plant height (PH), and five yield‐related traits, viz. grain weight per panicle (GWP), number of grains per panicle (GPP), 1000‐grain weight (TGW), seed set (SS) and number of panicles of per plant (PPP), were evaluated over 3 years. Quantitative trait loci (QTL) analysis was conducted using a likelihood ratio test based on stepwise regression. Forty‐six putative QTL distributed on 11 chromosomes were detected in more than one year. Remarkably, GWP of four CSSLs carrying positive yield QTL outperformed the recurrent parent ‘93‐11’ by more than 15%, in at least two environments. These results indicate that CSSLs are effective in identifying yield‐associated traits, and lines harbouring such QTL will be rich in resources for future molecular breeding programmes.     

Application of marker‐assisted backcross to introgress Bph3, Bph14 and Bph15 into an elite indica rice variety for improving its resistance to brown planthopper

To improve brown planthopper (Nilaparvata lugens Stål; BPH) resistance of an elite indica cultivar of South China, Hemeizhan (HMZ), we applied marker‐assisted backcross (MABC) to incorporate three BPH‐resistance genes (Bph3, Bph14 and Bph15) into the genetic background of HMZ. In the third backcross (BC₃) generation, we obtained near‐isogenic lines (Bph3‐NIL, Bph14‐NIL, Bph15‐NIL and Bph14 + Bph15‐NIL) with more than 96% recovery of recurrent parent genome, and pyramided lines (Bph3 + Bph14‐PYL, Bph3 + Bph15‐PYL and Bph3 + Bph14 + Bph15‐PYL) with more than 89% recovery of recurrent parent genome. These lines showed stronger resistance against BPH than HMZ at seedling and booting stages. The rank of resistance gene effect was Bph3 + Bph14 + Bph15 ≥ Bph3 + Bph15 ≥ Bph3 +Bph14 ≥ Bph14 + Bph15 ≥ Bph3 ≥ Bph15 ≥ Bph14 > none. Compared with HMZ, only Bph3 + Bph14 + Bph15‐PYL had a significant difference in yield per plant, and the lines carrying Bph3 had higher amylose contents, indicating that Bph3 was tightly linked to Wx^a allele. These improved lines are good intermediate sources of broad‐spectrum and durable BPH resistance to improve other indica cultivars. Our results demonstrate that MABC is a very efficient approach to improve BPH resistance of elite rice cultivar.     

Introgression of Xa4, Xa7 and Xa21 for resistance to bacterial blight in thermosensitive genetic male sterile rice (Oryza sativa L.) for the development of two-line hybrids

Bacterial blight (BB) of rice caused by X. oryzae pv. oryzae is a major production constraint in commercial hybrid rice production in the Philippines because most of the parental lines used in hybrid production do not carry resistance genes against the pathogen. In this study, three bacterial blight resistance genes, Xa4, Xa7 and Xa21, were introgressed to a temperature-sensitive genetic male sterile (TGMS1) line. A three-way cross of AR32-19-3-3/TGMS1//IRBB4/7 (PR36944) was made to produce 1,364 F₂ plants carrying various combinations of Xa4, Xa7 and Xa21. Individual plants were characterized for reaction to bacterial blight PXO61 (race 1), PXO86 (race 2), PXO99 (race 6) and pollen sterility. Of 144 F₂ plants demonstrating resistance against PXO61, PXO86 and PXO99, 22 exhibited highly resistant phenotypes with mean lesion lengths ranging from 0.37–2.97 cm. Analysis of disease reaction identified 20 potential TGMS F₂ plants containing Xa4, Xa7 and Xa21 while 78 plants with Xa4 + Xa7. Phenotypic and polymerase chain reaction (PCR) analyses confirmed PR36944-450, PR36944-473 and PR36944-700 as homozygous for Xa7 and Xa21 and highly resistant to all three Xoo races. Fertility of PR36944-450 and PR36944-700 was restored at permissive temperature in a growth chamber. BB-resistant TGMS lines should facilitate breeding two-line hybrids in the tropics.